Nitric oxide and polyamine pathway-dependent modulation of neutrophil free amino- and α-keto acid profiles or host defense capability

Summary. We have examined the effects of N_ω-nitro-L-arginine-methylester-hydrochloride [L-NAME; inhibitor of nitric oxide synthase], S-nitroso-N-acetyl-penicillamine [SNAP; nitric oxide donor], α-difluoro-methyl-ornithine [DFMO; inhibitor of ornithine decarboxylase] arginine or ornithine as well as the combination of arginine or ornithine with L-NAME, SNAP or DFMO on intracellular free amino- and α-keto acid profiles and the immune function markers superoxide anion and hydrogen peroxide generation as well as released myeloperoxidase activity in neutrophils (PMN). Although the underlying mechanisms still remain unclear, we believe from our results that nitric oxide as well as polyamine-dependent pathways are involved in the signal transmission of free radical molecule, beneficial nutritional therapy or maleficient pharmacological stress-induced alterations in PMN nutrient composition. Relevant changes in intragranulocyte free amino- and α-keto acid homeostasis and metabolism, especially, may be one of the determinants in PMN nutrition that positively or negatively influences and modulate neutrophil host defence capability and immunocompetence.     

Pathways involved in alanyl-glutamine-induced changes in neutrophil amino- and α-keto acid homeostasis or immunocompetence

Summary. We examined the effects of DON [glutamine-analogue and inhibitor of glutamine-requiring enzymes], alanyl-glutamine (regarding its role in neutrophil immunonutrition) and alanyl-glutamine combined with L-NAME, SNAP, DON, β-alanine and DFMO on neutrophil amino and α-keto acid concentrations or important neutrophil immune functions in order to establish whether an inhibitor of •NO-synthase [L-NAME], an •NO donor [SNAP], an analogue of taurine and a taurine transport antagonist [β-alanine], an inhibitor of ornithine-decarboxylase [DFMO] as well as DON could influence any of the alanyl-glutamine-induced effects. In summary, irrespective of which pharmacological, metabolism-inhibiting or receptor-mediated mechanisms were involved, our results showed that impairment of granulocytic glutamine uptake, modulation of intracellular glutamine metabolisation and/or de novo synthesis as well as a blockade of important glutamine-dependent metabolic processes may led to significant modifications of physiological and immunological functions of the affected cells.     

Effects of hypoxia on plasma amino acids of fetal sheep

Summary. Secondary amino acid disturbances from circulatory responses during hypoxia may cause problems in interpreting plasma amino acid profiles of sick babies investigated for possible inherited defects. Systematic studies to characterise them are difficult in man. We investigated the effects of hypoxia on plasma amino acids by studying 9 late gestation fetal sheep in utero during 11 one hour episodes of moderately severe isocapnic hypoxia. In 6 experiments, maternal plasma amino acids were also monitored. Fourteen fetal plasma amino acids increased significantly, with the largest proportionate changes in alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, ornithine and lysine. Maternal amino acids did not increase. Probable explanations were reflex peripheral vasoconstriction in skeletal muscle beds and decreased hepatic blood flow. The findings extend our knowledge of the fetal response to hypoxic stress, demonstrate the importance of skeletal muscle in branched-chain amino acid metabolism, and should help with interpretation of postnatal plasma amino acid disturbances. Received January 29, 1999, Accepted February 22, 1999     

Relationships between the sedative and hypnotic effects of intracerebroventricular administration of L-serine and its metabolites, pyruvate and the derivative amino acids contents in the neonatal chicks under acute stressful conditions

Summary. Intracerebroventricular (i.c.v.) injection of L-serine was shown to have sedative and hypnotic effects on neonatal chicks under acute stressful conditions. To clarify the central mechanism of these effects of L-serine, two experiments were done. First, we focused on the glycogenic pathway in which L-serine is converted into pyruvate and finally glucose. I.c.v. administration of pyruvate (0.84 μmol) did not induce any behavioral and endocrinological changes, while L-serine and glucose triggered sedative and hypnotic effects. Secondly, the relationship between the sedation by L-serine and the metabolism into other amino acids which have sedative effects was investigated in the telencephalon and diencephalon. In both brain areas, a dose-dependent increase was seen in L-serine, although other amino acids were not changed. In the present study, it was concluded that the sedative action of L-serine was not due to the action of its metabolite pyruvate, or to the action of other amino acids. Authors’ address: M. Furuse, PhD, Laboratory of Advanced Animal and Marine Bioresources, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, Fukuoka 812-8581, Japan     

Surprising insights that aren’t so surprising in the modeling of sulfur amino acid metabolism

Summary. The modeling of whole organism sulfur amino acid flux control has been aided in recent years by advancements in proteomics and mass spectroscopy-based metabolite analysis. The convergence of these two fields and their respective techniques, as demonstrated by a new study using yeast by Lafaye et al., has shown that researchers seeking to model whole cell/organism metabolism should give careful consideration to the relationships connecting enzyme concentration, enzyme activity, substrate concentration, and metabolic flux. In this paper, we outline some of the fundamental concepts for modeling sulfur amino acid metabolism and how they relate to our current understanding of mammalian sulfur amino acid metabolism.     

Indicators of l-arginine metabolism and cardiovascular risk factors A cross-sectional study in healthy middle-aged men

Summary. This study examines the relationship between traditional risk factors of coronary artery disease and indicators involved in the metabolism of l-arginine (plasma and urine l-arginine, plasma l-citrulline, serum creatinine and urine orotic acid). Our study population consisted of 40 healthy male volunteers aged between 35 and 55 years. We found an inverse association between serum creatinine and blood pressure, between plasma l-citrulline and blood pressure, as well as between urine l-arginine and blood pressure. We also found a positive association between plasma LDL-cholesterol and urine l-arginine and a negative correlation between plasma l-arginine and LDL-cholesterol. Orotic acid measured from urine was not associated with any of the indicators of l-arginine metabolism. Our results indicate that l-arginine metabolism is of profound significance for cardiovascular health. However, our study does not answer questions relating to causality. Further studies are needed to clarify the causal relationship between cardiovascular risk factors, especially elevated blood pressure and high LDL-cholesterol, and indicators of l-arginine metabolism. Received January 18, 1999     

Multi-layered network structure of amino acid (AA) metabolism characterized by each essential AA-deficient condition

Summary. The concentrations of free amino acids in plasma change coordinately and their profiles show distinctive features in various physiological conditions; however, their behavior can not always be explained by the conventional flow-based metabolic pathway network. In this study, we have revealed the interrelatedness of the plasma amino acids and inferred their network structure with threshold-test analysis and multilevel-digraph analysis methods using the plasma samples of rats which are fed diet deficient in single essential amino acid. In the inferred network, we could draw some interesting interrelations between plasma amino acids as follows: 1) Lysine is located at the top control level and has effects on almost all of the other plasma amino acids. 2) Threonine plays a role in a hub in the network, which has direct links to the most number of other amino acids. 3) Threonine and methionine are interrelated to each other and form a loop structure.     

Regulation of Growth Hormone (GH) secretion by different glutamate receptor subtypes in the rat

Summary. It has been firmly established that excitatory amino acids (EAAs), such as glutamate, are pivotal elements in the hypothalamic circuitry involved in the control of pituitary function. The actions of EAAs are mediated by different postsynaptic receptor subtypes, which include N-methyl D-aspartate (NMDA), kainate (KA), 2-amino-3-hydroxy-5 methyl-4-isoxazol propionic acid (AMPA) and metabotropic receptors. In this review, we summarize our experimental work on the role of EAA neurotransmission in the control of GH secretion in the rat. Detailed characterization of the effects of agonists and antagonists of glutamate receptors on GH release revealed that activation of NMDA, KA and AMPA receptors at different age-points resulted in clear-cut stimulation of GH secretion, although age- and sex-dependent differences were detected in the pattern of response to the different agonists. This stimulatory action was proven nitric oxide (NO)-dependent and not exerted at the pituitary level. In addition, evaluation of the role of hypothalamic GH-releasing hormone (GHRH) in the stimulatory action of NMDA by means of immunoneutralization of endogenous GHRH or destruction of GHRH producing neurons suggested the involvement of signals other than GHRH in this response. Further, evidence was obtained on the modulation of the EAA system by gonadal factors, and on the physiological relevance of EAA pathways in the regulation of pulsatile GH release. In conclusion, our data using the rat as animal model provide evidence for a pivotal role of glutamate pathways in the regulation of GH secretion throughout the life-span. Received May 5, 1999, Accepted July 28, 1999     

Which mechanisms are involved in taurine-dependent granulocytic immune response or amino- and α-keto acid homeostasis?

We examined the effects of beta-alanine (taurine analogue and taurine transport antagonist), taurine (regarding its role in neutrophil (PMN) immunonutrition) and taurine combined either with L-NAME (inhibitor of *NO-synthase), SNAP (*NO donor), DON (glutamine-analogue and inhibitor of glutamine-requiring enzymes), DFMO (inhibitor of ornithine-decarboxylase) and beta-alanine on neutrophil amino- and alpha-keto acid profiles or important PMN immune functions in order to establish whether taurine transport-, nitric oxide-, glutamine- or ornithine-dependent mechanisms are involved in any of the taurine-induced effects. According to the present findings, the taurine-mediated effect appears to be based primarily on a modulation of important transmembraneous transport mechanisms and only secondarily on directly or indirectly induced modifications in intragranulocytic amino- and alpha-keto acid homoeostasis or metabolism. Although a direct relation to the parallel observed immunological modifications can only be presumed, these results show very clearly that compositional modifications in the free intragranulocytic amino- and alpha keto-acid pools coinciding with changes in intragranulocytic taurine levels are relevant metabolic determinants that can significantly influence the magnitude and quality of the granulocytic immune response.     

When quinones meet amino acids: chemical, physical and biological consequences

Summary. Quinones and amino acids are usually compartmentally separated in living systems, however there are several junctions in which they meet, react and influence. It occurs mainly in wounded, cut or crushed plant material during harvest, ensiling or disintegrating cells. Diffusing polyphenols are oxidized by polyphenol oxidases (PPOs) to quinonic compounds, which associate reversibly or irreversibly with amino acids and proteins. The reaction takes place with the free nucleophilic functional groups such as sulfhydryl, amine, amide, indole and imidazole substituents. It results in imine formation, in 1,4-Michael addition via nitrogen or sulphur and in Strecker degradation forming aldehydes. The formation and activity of quinone–amino acids conjugates influences the colour, taste, and aroma of foods. Physical and physiological phenomena such as browning of foods, discoloration of plants during processing, alteration of solubility and digestibility, formation of humic substances, germicidal activity, cytotoxicity and more occur when quinones from disintegrating cells meet amino acids. The mechanisms of toxicity and the pathways by which PCBs may be activated and act as a cancer initiator include oxidation to the corresponding quinones and reaction with amino acids or peptides. Sclerotization of insect cuticle is a biochemical process involving also the reaction between quinones and amino acid derivatives.     

Relationship of taurine and other amino acids in plasma and in neutrophils of septic trauma patients

Summary. Recently, an interdependency of plasma taurine and other amino acids as well as metabolic and clinical variables implicating therapeutic options was reported. This result may be an indication that plasma taurine levels are directly related to intracellular levels. Therefore, the aim of this study was to analyse the possible relationship between taurine levels in plasma and in neutrophils, the relationship to other amino acids, and variables quantifying metabolic impairment and severity of sepsis in multiple trauma patients developing sepsis. After multiple trauma taurine decreased significantly in plasma in thirty-two patients as well as within the neutrophil and does not recover in sepsis. Lower individual levels in the neutrophil did not follow lower individual levels in plasma and no correlation of taurine in plasma and in the neutrophils could be observed. In sepsis, only plasma showed an interdependency of taurine, aspartate, and glutamate. No association between taurine plasma or intracellular levels and SOFA score as indicator for severity of sepsis or metabolic variables was observed. After multiple trauma and in sepsis, taurine uptake in cells (which is regulated in different ways), and intracellular taurine (which serves e.g. as an osmolyte) can be influenced. Therefore a prediction of the neutrophil taurine pool seems not fully possible from taurine plasma levels. Intracellular taurine has some unique properties explaining the missing interdependency despite some similarities in osmoregulation and metabolic interactions to other amino acids. The association of taurine, aspartate, and glutamate in plasma cannot be simply transferred to the neutrophils intracellular level. The clinical meaning of the plasma correlation remains unclear. A dependency of plasma and neutrophil taurine to severity of sepsis and to metabolic variables seems not possible because of the multifactorial pathophysiology of sepsis.     

Biochemical composition of algivorous freshwater ciliates: you are not what you eat

The focus of our study was to determine whether the biochemical composition of two algivorous ciliates, both fed the same alga, resembles that of their diet. By comparing both ciliated protozoa we intended to identify species-specific differences in the metabolic features of these ciliates. Carbon- and cell-specific concentrations of fatty acids and essential amino acids were investigated for the ciliates Balanion planctonicum and Urotricha farcta grown on the cryptomonad Cryptomonas phaseolus. Stepwise discriminant analyses (SDA) indicated differences in the biochemical composition between ciliates and their diet and between the two ciliated protozoa. Carbon-specific fatty acid concentrations were usually higher in the ciliates than in their diet, especially concentrations of monounsaturated and some polyunsaturated fatty acids. Except for tryptophan, valine, and lysine, amino acid concentrations were higher in the ciliates than in C. phaseolus. Furthermore, differences in the polyunsaturated fatty acids accounted for the largest discrepancies between the two ciliated protozoa. The higher concentrations in the ciliates compared to their diet suggest that these species are capable of efficiently ingesting, assimilating or possibly synthesizing some fatty acids and amino acids. We conclude that dietary fatty acid and amino acid composition influences the composition of the two ciliated protozoa to a minor extent, and that species-specific differences in fatty acid and amino acid metabolism may be more important determinants of the biochemical composition of the studied ciliates. Moreover, the metabolism of polyunsaturated fatty acids seems to differ more profoundly between the two ciliated protozoa than the metabolism of other fatty acid classes or amino acids.     

Cysteine dioxygenase and γ-glutamylcysteine synthetase activities in primary cultured hepatocytes respond to sulfur amino acid supplementation in a reciprocal manner

Summary. Hepatocytes were cultured for 3 days as spheroids (aggregates) or as monolayers in basal medium and in sulfur amino acid-supplemented media. Cultured hepatocytes had low levels of cysteine dioxygenase (CDO) activity and normal levels of γ-glutamylcysteine synthetase (GCS) and cysteinesulfinate decarboxylase (CSDC) activities compared to freshly isolated cells. CDO activity increased and GCS activity decreased in a dose-response manner in cells cultured in either methionine- or cysteine-supplemented media. CSDC activity was not significantly affected by methionine supplementation. Changes in CDO and GCS were associated with changes in cysteine catabolism to taurine plus sulfate and in synthesis of glutathione, respectively. These responses are similar to those observed in liver of intact rats fed diets supplemented with sulfur amino acids. A near-maximal response of CDO or GCS activity was observed when the medium contained 1.0 mmol/L of methionine plus cyst(e)ine. Changes in CDO and GCS activities did not appear to be mediated by changes in the intracellular glutathione concentration. Cultured hepatocytes offer a useful model for further studies of cysteine metabolism and its regulation in response to sulfur amino acid availability. Received June 2, 1999/Accepted September 16, 1999     

Influence of nitric oxide synthase activity on amino acid concentration in the quinolinate lesioned rat striatum: A microdialysis and histochemical study

Summary. The influence of nitric oxide synthase (NOS) activity on the KCl-evoked amino acid concentrations was investigated by in vivo microdialysis in the striatum in a rat model of excitotoxic lesion. Basal microdialysate levels of amino acids decreased during the quinolinic acid-induced neurodegeneration process, except for glutamine that increased initially and returned to control values 30 days after quinolinic acid exposure. KCl-evoked increase of extracellular amino acid concentration was reduced due to NOS activity in the striatum of both controls and lesioned animals, except for 120 days after quinolinic acid injection. These changes of amino acid concentrations in microdialysates correlated with the known biochemistry of the consecutive domineered cell types during the lesion process as revealed by histochemistry for NOS, NADPH-diaphorase, GFAP and isolectin B4. The present data provide direct evidence that NOS activity can modulate extracellular amino acid concentrations in the striatum not only under physiological conditions, but also during a pharmacologically induced lesion process and, thus, suggests that nitric oxide affects neurodegeneration via this pathway. Received October 20, 1999; Accepted February 25, 2000     

Amino acid transport in the renal proximal tubule

Summary. In the kidney the proximal tubule is responsible for the uptake of amino acids. This occurs via a variety of functionally and structurally different amino acid transporters located in the luminal and basolateral membrane. Some of these transporters show an ion-dependence (e.g. Na⁺, Cl⁻ and K⁺) or use an H⁺-gradient to drive transport. Only a few amino acid transporters have been cloned or functionally characterized in detail so far and their structure is known, while little is known about a majority of amino acid transporters. Only few attempts have been untertaken looking at the regulation of amino acid transport. We summarized more recent information on amino acid transport in the renal proximal tubule emphasizing functional and regulatory aspects. Received August 8, 1999; Accepted April 20, 2000     

Indispensable amino acid concentrations decrease in tissues of stomachless fish,common carp in response to free amino acid- or peptide-based diets

Summary. The premise that free amino acid or dipeptide based diets will resolve the nutritional inadequacy of formulated feeds for larval and juvenile fish and improve utilization of nitrogen in comparison to protein-based diets was tested in stomachless fish, common carp (Cyprinus carpio L.) larvae. We examined the postprandial whole body free amino acid (FAA) pool in fish that were offered a FAA mixture based diet for the duration of 2 or 4 weeks. We found that the total amount and all indispensable amino acids concentrations in the whole body decreased after a meal. We then fed juvenile carp with dietary amino acids provided in the FAA, dipeptide (PP), or protein (live feed organisms; brine shrimp Artemia salina nauplii, AS) forms. Histidine concentrations in the whole fish body increased in all dietary groups after feeding whereas all other indispensable amino acids decreased in FAA and PP groups in comparison to the AS group. Taurine appears to be the major osmotic pressure balancing free amino acid in larval freshwater fish which may indicate a conditional requirement. We present the first evidence in larval fish that in response to synthetic FAA and PP diets, the whole body indispensable free AA concentrations decreased after feeding. This study shows that amino acids given entirely as FAA or PP cannot sustain stomachless larval fish growth, and may result in depletion of body indispensable AA and most of dispensable AA. The understanding of these responses will determine necessary changes in diet formulations that prevent accelerated excretion of amino acids without protein synthesis.     

Plasma arginine correlations in trauma and sepsis

Summary. Arginine (ARG) is an amino acid (AA) with unique properties and with a key-role in the metabolic, immune and reparative response to trauma and sepsis. This study has been performed to characterize the correlations between plasma levels of ARG, of other AA and of multiple metabolic variables in trauma and sepsis. Two-hundred and sixty-three plasma amino-acidograms with a large series of additional biochemical and blood variables were obtained consecutively in 9 trauma patients who developed sepsis, undergoing total parenteral nutrition with dextrose, fat and a mixed AA solution containing 10.4% arginine. ARG was low soon after trauma, then it increased with increasing distance from trauma and with the development of sepsis. ARG was also directly related to the AA infusion rate (AAIR) and for any given AAIR, was lower after trauma than after the development of sepsis. ARG was also related directly to the plasma levels of most of the other AA, the best correlation being that with lysine (r² = 0.81, p < 0.001). These correlations were often shifted downwards (showing lower ARG for any given level of the other AA) in measurements performed after trauma, compared to those performed after development of sepsis; this effect was more pronounced for the correlations with branched chain AA. Correlations between ARG and non-AA variables were not particularly relevant. The best simultaneous correlates of ARG, among variables involved in plasma ARG availability, were citrulline level, AAIR and urinary 3-methylhistidine excretion (accounting for the effect of endogenous proteolysis) (multiple r² = 0.70, p < 0.001). Plasma ornithine (ORN), the AA more specifically linked to ARG metabolism, correlated with AAIR better than ARG and, for any given AAIR, was lower after trauma than after the development of sepsis. Correlations of ORN with other AA levels were poorer than those found for ARG, however ORN was directly related to white blood cell and platelet count, fibrinogen, transferrin, cholesterol and many AA clearances. These data show that changes in ARG in trauma and sepsis are correlated with changes in other AA and, within these correlations, reconfirm a tendency to lower ARG in trauma compared to sepsis. The strong correlation with lysine warrants a deeper assessment of the practical implications of interdependency between these two AA. The data also suggest that changes in plasma ORN in trauma and sepsis may reflect adequacy of AA substrate to support acute-phase and other synthetic processes.     

Consequences of renal mass reduction on amino acid and biogenic amine levels in nephrectomized mice

Summary. Amino acid and biogenic amine changes were investigated in nephrectomized mice ten days postsurgery. Uremic mice exhibited changes in amino acid concentrations in plasma, urine and brain. Particularly plasma methionine, citrulline and arginine levels were significantly enhanced in nephrectomized mice compared to controls whereas serine was decreased. Urinary excretion of methionine, citrulline and alanine was higher in nephrectomized mice compared to controls whereas many amino acids were increased in brain of nephrectomized mice. Brain and urinary amino acid changes were more pronounced in the 75% than in the 50% nephrectomized mice. Brain norepinephrine and dopamine and its metabolites 3,4-dihydroxyphenylacetic acid and homovanillic acid were significantly increased whereas serotonin was decreased comparing the 75% nephrectomized mice to the sham-operated mice. This study demonstrates that at very early stages of renal insufficiency, specific amino acid and biogenic amine changes occur in plasma, urine and brain. These alterations might depend qualitatively and quantitatively on the degree of functional renal mass reduction. Received April 5, 1999     

Two new reactive targets of 2,5-hexanedione in vitro – beta-alanine and glycine

Summary. In this study, we found that two amino acids reacted with 2,5-hexanedione to form new reaction products in vitro, respectively. In the reaction of beta-alanine and 2,5-hexanedione, a reaction product was obtained and analyses of obtained results showed it was 3-(2,5-dimethyl-1H-pyrrol-1-yl)propanoic acid; in the reaction of glycine and 2,5-hexanedione, a reaction product was also obtained and analyses showed it was (2,5-dimethyl-1H-pyrrol-1-yl)acetic acid. Two reaction products were found to be oxidized easily; in addition, the latter was more easily to be oxidized than the former in the air. Our discoveries demonstrated that reactions between amino acids and 2,5-hexanedione could exist possibly in vitro. At present, it is clear that 2,5-hexanedione causes either axon atrophy or swelling, but the underlying molecular mechanism is still unclear. Since both beta-alanine and glycine are considered as neurotransmitter in the central nervous system, the reaction products remain to be identified in vivo.     

Free amino acid and dipeptide changes in the body fluids from Alzheimer’s disease subjects

Summary. Our aim was to determine changes in free amino acid (FAA) and dipeptide (DP) concentrations in probable Alzheimer’s disease (pAD) subjects compared with control (CT) subjects using liquid chromatography and electrospray ionization tandem mass spectrometry (LCMS²). We recruited gender- and age-matched study participants based on neurological and neuropsychological assessments. We measured FAAs and DPs in cerebrospinal fluid (CSF), plasma and urine using LCMS² with selected reaction monitoring (SRM). Imidazole-containing FAAs (histidine, methyl-histidine), catecholamines (L-DOPA and dopamine), citrulline, ornithine, glycine and antioxidant DPs (carnosine and anserine) accounted for the major changes between CT and pAD. Carnosine levels were significantly lower in pAD (328.4 ± 91.31 nmol/dl) than in CT plasma (654.23 ± 100.61 nmol/dl). In contrast, L-DOPA levels were higher in pAD (1400.84 ± 253.68) than CT (513.10 ± 121.61 nmol/dl) plasma. These data underscore the importance of FAA and DP metabolism in the pathogenesis of AD. Since our data show changes in antioxidants, neurotransmitters and their precursors, or FAA associated with urea metabolism in pAD compared with CT, we propose that manipulation of these metabolic pathways may be important in preventing AD progression.